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蚂蚁淘在线 / 品牌中心 / Everest Biotech / Everest Biotech/Cyclin D1免疫组织化学试剂盒10张幻灯片/3100050105/10张幻灯片

Everest Biotech/Cyclin D1免疫组织化学试剂盒10张幻灯片/3100050105/10张幻灯片

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货号：3100050105

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Duringthecellcycleofmostsomaticcells,DNAsynthesis(S-phase)andmitosis(M-phase)areseparatedbytwogapphases(G1andG2)ofvaryingduration.Regulationofcellcycleprogressionineukaryoticcellsdependsontheexpressionofcyclinproteins.Membersofthecyclinfamilyofproteinscombinewithacyclindependentkinases(CDKs)subunittoformtheactivekinase,whichinitiatesG2toMandG1toStransition.ThelatterarecontrolledbycyclinstermedG1cyclins,whichcommitthecelltoDNAreplication.AtleastfivecandidateG1-phasecyclins,termedcyclinsC,D1,D2,D3,andEhavebeenidentifiedinmammaliancells.D-typecyclinsareinducedduringtheG1phaseofthemammaliancellcycleinresponsetoavarietyofmitogenicgrowthfactors.Onceinduced,theD-typecyclinsaccumulateincomplexeswithCDKs,whosekinaseactivityisthoughttobenecessaryfordrivingcellsintoSphase.ThemajorcatalyticpartnersoftheD-typecyclinsareCDK4andCDK6,butatleastsomeD-typecyclinsalsointeractwithotherCDKs,includingCDK2andCDK5.CyclinD1-andD2-associatedCDK4and/orCDK6kinaseactivitieshavebeendetectedinmid-G1,priortotheactivationofanyotherknownCDK,andtheyculminateinlateG1phase.AmplificationofthecyclinD1genehasbeenobservedinasignificantpercentageofcancers,includingbreast,squamous,andesophagealcarcinomas.

Imageshowsimmunohistochemicalstainingofparaffin‐embeddedhumanbreastadenocarcinomaxenografttumorsectionstainedwithCyclinD1antibodyusingtheEtonBio"sCyclinD1IHCKit.CyclinD1(darkbrown)displaysanuclearlocalizationpatternwhichcorrelatesitsfunctioninregulatingcycleprogression(20X,counterstainedwithhematoxylin).

KitContents

Reagentsprovidedinthekit

Thenumberoftestsisbasedontheuseof200μLeachofready-to-usereagentperslide.

PositiveControlSlide

•positivecontrolslide

EquilibriumBuffer

•EquilibriumBuffer

•Ready-to-usereagent

BlockingBuffer

•10XNon-specificblockingbuffer

•Diluteat1:10usingdistilledwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.

RabbitcyclinD1antibody

•Rabbitanti-cyclinD1antibody

•DiluteinAntibodyDiluentsimmediatelybeforeuse(recommenduseat1:50dilution).

AntibodyDiluent

•AntibodyDilutent

•Ready-to-usereagent

WashBuffer(20x)

•TrisbufferedsalinewithTween20(pH7.6)

•Diluteat1:20usingdistilledordeionizedwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.

RabbitHRPPolymer

•RabbitHRPPolymer

•Ready-to-usereagent

DABsubstratebuffer

•10XDABsubstratebuffer

•Beforeuse,add100μL10XDABsubstratebufferto1mLofdistilledordeionizedwatertomake1XDABsubstratebuffer.

HydrogenPeroxide(H₂O₂)forDABsubstratebuffer

•0.3%HydrogenPeroxidesolution

•Beforeuse,add15μLH₂O₂to1mLof1XDABsubstratebuffer.

DABChromogen

•Diaminobenzedinetetrahydrochloride(DAB)substratesolution

•DABSubstrateSolution

Materialsrequiredbutnotincludedinthekit

Reagents:

•Xylene

•Ethanol

•EndogenousPeroxidaseBlockingSolution(3%HydrogenPeroxide)

•Hematoxylin

•Mountingmedia

•Distilledordeionizedwater

•AntigenRetrievalBuffer(10X)0.1MCitrateBuffer(pH6.0)Diluteat1:10using

LabEquipment:

•Steamerormicrowaveoven(forantigenretrieval)

•Generallabequipmentforimmuno-histostainingsuchasslideracks,stainingjars,coverslips,timer,Pipettes,etc.Microscopeequipmentandaccessories

StorageandstABIlity

StoreIHCKitsat2‐8°C.Thekitisstableforsixmonthsat4°C.

Donotuseafterexpirationdate.

Precautions

Takereasonableprecautionswhenhandlingreagents.UsedisposablegloveswhenhandlingsUSPectedcarcinogensortoxicmaterials(examples:DAB,xyleneandH₂O₂).Unusedsolutionshouldbedisposedaccordingtoapplicablelocal,stateandfederalregulations.

TheCyclinD1ImmunohistostainingKithasbeendesignedforthestainingoftissuesthathavebeenfixed(usuallyinneutralbufferedformalin)andsubsequentlyembeddedinparaffinbeforesectioning.Thisprotocolisrecommendedasastartingpoint.Wheneverusinganewantibodyorimmunohistochemistrykit,optimizationbytheindividualend-usermayberequired.

Note:

•Donotallowspecimenstodryduringthestainingprocedure.Specimendryingmaycauseincreasednon-specificstainingandbackground.

•Sometissuemayneedtobaketoremoveover-coveredparaffinpriortotheprocedure.Ifneeded,bakeat55-60°Cfor30minutes.

I.Deparaffinizationandrehydration

Priortostaining,tissuesectionsmustbedeparaffinizedandrehydrated.Incompleteremovalofparaffincancausepoorstainingofthesection.

Immerseslidesinxyleneandincubatefor5minutes.Repeattwicewithfreshxyleneforanother5minuteseach.
Immerseslidesin100%ethanolfor5minutes,andfollowwithimmersionin95%,75%and50%ethanolfor3minuteseach.
Rinseslideswithdistilledwaterfor5minutes;keepinwateruntilreadytoperformantigenretrieval.

II.Heatinducedantigenretrieval(HIAR)

Mostformalin-fixedtissuerequiresanantigenretrievalstepbeforeimmunohistochemicalstainingcanproceed.Heatinducedantigenretrievalcanbeperformedusingasteamer,pressurecooker,oramicrowave.Theretrievaltimewritteninthisprotocolisbasedonusingaretrievalsteamer.Theheatingtimemayneedtobeadjustedifyouuseadifferentdeviceandmethod.

1.Fillplasticcoplinjar/containerwithAntigenRetrievalBuffer(0.01MCitrateBuffer,pH6.0,notincludedinthekits).

PrepareStockSolution:

0.1MSodiumCitrate--20.5mL

0.1MCitricAcid--4.5mL

Adddistilledwaterto250mL

2.Placethecoplinjar/containerinsteamerwithlid.
3.Turnonsteamerandpreheatto90‐100°C.Carefullyputslidesintothecoplinjar/containerandsteamfor40min(95‐100°C).
4.Turnoffthesteamer,removethecoplinjar,placeatroomtemperatureandallowslidestocoolfor20min.Keepthejarcoveredallthetime.
5.Rinseslidebyincubationofslideindistilledwaterfor3minutes.Repeatthissteptwiceandbeginstainingprocedure.

III.Stainingprocedure

PeroxidaseBlockingisoptional.Ifnonon-specificstainingisobserved,skipthesestepsandgotostep3.

BlockingofEndogenousPeroxidase

Note:PeroxidaseBlockingisoptional.Ifnonon-specificstainingisobserved,skipthesestepsandgotostep3.

1.Tapoffexcesswater.DrawacirclearoundthespecimenontheslidewithPAPpen(notincludedinthekit.Alternatively,foldedKimwipescouldbeusedtobrieflyblotthewateraroundthespecimen.Repeatthisblotstepeachtimebeforeaddreagentonslide).Apply200μlormoreofPeroxidaseBlockingSolution(notincludedinthekit)sufficienttocoverspecimen,andincubatefor5minutes.

2.Rinseslidebyincubationofslideindistilledwaterfor3minutes.Repeatthissteptwice.

3.RinseslidebyincubationslideinPBSfor3minutes.

BlockingofNon-specificbinding

4.TapoffexcessPBS.(IfthePeroxidaseBlockingstepisskipped,drawacirclearoundthespecimenontheslidewithPAPpenorusingtheedgeoffoldedKimwipestoquicklyblotthewateraroundthespecimen).Apply200μl1XBlockingBufferimmediatelytocoverspecimenandincubateinamoistchamberfornomorethan10minutes

Note:10Xblockingbuffermayformprecipitatesat4°C.Completelydissolvetheprecipitatesbeforemakingworkingsolution

5.RinseslidebyincubationslideinPBSfor3minutes.

PrimaryAntibody

6.TapoffexcessPBS.Apply200μlEquilibriumBufferimmediatelytocoverspecimenandincubateinamoistchamberfor30minutes

7.TapoffexcessEquilibriumBuffer.Apply200μldilutedanti‐CyclinD1antibody(recommend1:50dilutioninAntibodyDiluent)tocoverspecimenimmediatelyandincubateinamoistchamberovernightat4°C.

8.Rinseslidebyincubationin0.5-2mLWashBufferfor3minutes.Repeatthissteptwicewithfreshbuffer.

9.RinseslidebyincubationofslideinPBSfor3minutes.

Secondary/HRPConjugates

10.TapoffexcessPBS.Apply200μlRabbitHRPPolymerimmediatelytocoverspecimenandincubateinamoistchamberfor60minutes.

11.Rinseslidebyincubationin0.5-2mLWashBufferfor3minutes.Repeatthissteptwicewithfreshbuffer.

12.RinseslidebyincubationinwithPBSfor3minutes.

DABChromogen
13.TapoffexcessPBS.ApplyenoughDABSubstrateSolutiontocoverspecimenimmediatly.Checkdarkbrowncolordevelopmentundermicroscopeandincubateuntildesiredstainintensitydevelops.

Tomake1mLDABSubstrateSolution,mixthefollowingreagents:

DistilledWater--860μL

10XDABsubstratebuffer--100μL

0.3%HydrogenPeroxidesolution--15μL

DABChromogen--25μL

14.Rinseslideintapwaterfor3minutes.

Counterstaining

15.Ifdesired,completecounterstain(Seeinstructionforhematoxylincounterstaining).Rinseintapwatertoclear.

Mounting

16.Immerseslidesin70%,80%,95%Ethanolfor2minuteseach,and100%Ethanolfor10minutestwicefollowedbyXylenefor5minutestwice.

17.Dryandmountslides.

IV.InstructionforHematoxylincounterstaining

Immerseslidesinhematoxylinsolution.Incubatefor30secondsto5minutes,dependingonthestrengthofhematoxylinused.
RinsetoclearwithtapwaterandcontinuebydehydrationfromStep14.

Problems	PossIBLeCauses	Solutions
Overstaining	1.Toolongincubationtimeofprimaryantibody,ortoohightemperaturewhendoingstaining 2.ToolongincubationtimeofDABsubstrate. 3.Slidedriedduringstainingprocess	•Dependingontissuesections,theincubationtimeofprimaryantibodycanbereducedto2hours;Checktheroomtemperaturerangeisat20-250Cwhendoingstaining. •ReduceincubationtimeofDABsubstrate •Avoidsectionstodryduringstainingprocess.
Weakornostaining	1.Incompleteremovalofparaffin 2.Tissuesover‐fixation 3.Notefficientantigenretrieval 4.Reagentsnotusedinproperorderoromittedsteps 5.Expiredantibodyorreagents	•Deparaffinizesectionslongerorchangetofreshxylene;sometissuearraymayneedtobaketo removeover‐coveredparaffin. •Increasingtheconcentrationofprimaryantibodyto1:40;ifthisdoesnotwork,reducedurationof post‐fixation. •Adjustantigenretrievaltimebasedonthesettingforsectionfixationandretrievaldeviceused. •Reviewnotesandprocedureused. •Checkkitexpirationdatesandkitstorageconditions
Highbackground	1Sectionsdriedduringstainingprocess 2Slidenotrinsedthoroughly 3Antigenover‐retrieval	•Donotallowsectionstodryduringstainingprocess;usehumidcontainerduringincubation withprimaryantibody. •Usefreshsolutioninbufferjars;rinseatleastthreetimesbetweensteps. •Optimizeantigenretrievaltimeifyouusedmicrowaveorpressurecookerforretrieval.

Everest Biotech的山羊多克隆抗体以Everest Elite Grade或Aspiring Grade产品的形式发布，这取决于它们的测试性能和对靶蛋白的了解程度。Everest Biotech提供了圣克鲁斯多克隆产品系列的高质量替代品精英级抗体保证按数据表上的规定工作，或退款。肽ELISA数据显示它们可以识别免疫肽。Western印迹数据显示生理相关组织或细胞系上的正确大小带。IHC / IF数据和特定出版物（如果有）。100％满意保证也可批量提供给化验开发人员有抱负的抗体快速将市场上几乎没有或没有商业抗体可用且在文献中几乎没有表达数据的靶标引入市场。由肽ELISA数据支持。背景低但带大小错误或在可用于测试的常见细胞系和组织范围内没有信号。以大幅降低的价格出售，但我们不确定他们会在任何应用中识别天然蛋白质。100％满意保证奖励分享显示抗体成功发挥作用的数据-将您的数据提交到support@everestbiotech.com可获得您选择的免费抗体。免疫肽产生抗体的肽可用于封闭实验以100µg冻干肽的形式出售

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